Clostridium perfringens-like strains whose taxonomic position is uncertain were examined in detail. The lecithinases of these strains exhibited less avidity to the alpha-antitoxin of C. perfringens than did the C. perfringens lecithinase. On the basis of a computer analysis, the C. perfringens-like strains were grouped into two phenons, I and 111, both of which are distinctly separable from C. perfringens (phenon 11). Strains previously identified by us as belonging to Clostridium paraperfringens Nakamura et al. 1970, including strain G (= ATCC 27639), here designated as the type strain of C. paraperfringens, were found to belong t o phenon I, and the strains of phenon I11 are regarded as constituting a new species, for which we propose the name Clostridium absonum. The type strain of C. absonum is HA-7103 (= ATCC 27555). The main characters differentiating C. absonum from C. perfringens are as follows: C. absonum produces a lecithinase which exhibits extremely low avidity t o C. perfringens alpha-antitoxin, rapidly ferments salicin, does not ferment raffinose, and does not liquefy 10% gelatin; the main characters of C. absonum which differentiate it from phenon I strains are: larger cell width, a distinctly stronger lecithinase reaction, liquefaction of 2% gelatin, production of butanol, and weak toxicity for mice. Deoxyribonucleic acid (DNAFDNA homology studies of phenons I, 11, and I11 confirmed the validity of the above-mentioned groupings. A computer analysis of other saccharolytic clostridia revealed that C. butyricum, C. acetobutyricurn, and C. multifermentans are associated by similarity values higher than 90% and that C. septicum and C. chauvoei constitute separate, but closely related, taxa.In a previous communication, Nakamura et al. (15) proposed a new species, Clostridium paraperfringens, for strains isolated from a case of gas gangrene, from normal human intestines, and from soil samples. A type strain was not designated for this new species. The lecithinases produced by most of these C. perfn'ngens-like strains on half-antitoxin Nagler agar (26) were completely suppressed by an excessive amount of C. perfringens antitoxin although they exhibited less avidity t o C. perfringens alphaantitoxin than did the lecithinase of C. perfringens. These strains were nontoxic. Later, we encountered another group of C. perfringenslike strains which were toxigenic. The present work was carried out to elucidate the taxonomic relationship of the above-mentioned groups of C. perfringens-like strains t o C.perfringens as well as to other saccharolytic clostridia by the use of numerical taxonomy, to detect their genetic relatedness by deoxyribonucleic acid (DNA)-DNA homology analysis, and to designate the type strain of C. paraperfringens. Because no computer analysis has been performed on clostridia to date, we also considered it worthwhile t o examine a number of saccharolytic strains by this method, particularly those belonging to C. septicum and C.chauvoei.
THE taxonomic relationship between Clostridium bifermentans and C. sordellii has been well reviewed by Brooks and Epps (1958) and was subsequently studied by Nishida, Tamai and Yamagishi (1964). In view of Novotonys recent claim (1969) that both species should be classified on the basis of sugar components in the cell walls, and because of the similarity of both species in their cultural and biochemical properties, the problem was reinvestigated in the present study by means of numerical taxonomy and consideration of DNA-DNA homology, thermostability of homologous and heterologous duplexes, and analysis of sugar components in the cell walls. MATERIALS AND METHODSStrains used. The sources and designations of the strains are shown in table I.C. sordellii strain 82 was obtained from the Department of Bacteriology, Leeds University, in 1959; at that time, the strain was non-toxigenic and urease negative. The subsequent subculture of this strain stocked in cooked-meat broth is designated as strain 82-Kanazawa (82-KZ) in the present text. Strains 82-SJ2, 82-SJ3, and 82-SJ4 are the substrains of C. sordelZii strain 82 stocked in Leeds University by single-cell isolation (Huang, 1959). A culture of C. sordellii strain 82 was also received from Dr A. R. Prbvot, Pasteur Institute, Paris, in 1963; this strain is still toxigenic and urease positive and is designated as C. sordellii no. 8ZPasteur (82-P). C. sordelZii no. 7222R/100 is a non-toxigenic substrain obtained by heating the toxigenic C. sordellii strain 7222R at 100°C for 30 min. C. sordellii no. 1734/90 is another non-toxigenic substrain obtained by heating a toxigenic strain 1734 at 90°C for 30 min. (Huang, Tamai and Nishida, 1965).Numerical taxonomy. The properties used in the characterisation of the organisms were previously described by Nakamura et al. (1973). The substrates used for tests of deaminase activity are alanine, aspartate, arginine, glutamate, leucine, methionine, phenylalanine, serine, threonine, and valine. The number of features scored is 162. Procedures for the determination of the strain characteristics, similarity value (S-value), coding of data, and analysis of the S-value have been described previously (Nakamura et al., 1973).Determination of sugar components of cell walls. Paper chromatography with Toyo filter-paper no. 51 was used according to the method described by Cummins and Johnson (1971). The bacterial cells were cultured in PY medium containing 1 % fructose (PYF medium). The PY medium consisted (w/v) of proteose peptone (Difco No. 2) 2%, yeast extract (Daigo, Tokyo, Japan) 03%, NaCl 03%, and sodium thioglycollate 0.1 %. The culture was harvested usually in the late logarithmic phase.Growth inhibition by mannose. Volumes (0-1 ml) of 12-h cultures grown in the PY medium with 0.5% fructose were transferred into 10-ml volumes of the PY medium with or without 1 % mannose and incubated at 37°C. Bacterial growth was measured for optical density at Preparation of DNA. The cells were grown in the PYF medium. For preparation of 3H-labelled DN...
Twenty-seven of 37 non-toxigenic, urease-negative strains originally identified as Clostridium bifermentans that were isolated in the Antarctic are reidentified as C. sordellii by the tests for DNA-DNA homology, by the absence of mannose in the cell wall, and by growth inhibition of mannose. The test for cell wall sugar components of urease-negative and -positive strains of C. sordellii revealed that glucose, mannose, and rhamnose could not be detected in any of eight urease-negative strains used by galactose was detectable in seven of the eight strains and that glucose or galactose or both of the two sugars were present in the urease-positive strains tested.
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